Coronaviruses have demonstrated the capacity for host-species switching, both in natural epidemics, such as SARS, and in the laboratory in cells from different species following passage, recombinant swapping of the spike protein, or following electroporation of genome RNA. Like other plus-strand RNA viruses, coronaviruses replicate in the host cell cytoplasm in association with modified cellular membranes. Coronaviruses cause profound modifications of cytoplasmic membranes, inducing a reticulovesicular network containing double- membrane vesicles as putative sites of viral RNA synthesis, also known as replication complexes. However, the viral and cellular determinants of replication complex formation and function are not known. In addition, infection with the coronavirus mouse hepatitis virus (MHV) is associated with continuous membrane ruffling and internalization, but the mechanisms and role of membrane ruffling in coronavirus replication is not known. The overall goals of the proposed research program is to elucidate the conserved and unique viral and cellular proteins, membranes, and pathways that mediate coronavirus induced cell membrane modifications and replication complex formation and establish the requirements for the membrane modifications in coronavirus replication. The three integrated aims of this proposal will use MHV and SARS-CoV in replication, cell imaging, proteomic and biochemical experiments to define the cellular and viral components of replication complexes, and establish the mechanisms and role of membrane ruffling in coronavirus replication. The results of these studies will identify new virus-host interactions that may be critical in the ability of coronaviruses to establish replication in multiple cell types and move between species. In addition the studies will likely define new viral targets for studies of virus host range and interference with virus replication. Finally the experiments may elucidate new pathways in host cell biology and interaction with intracellular pathogens. PUBLIC HEALTH RELEVANCE: Coronaviruses are RNA viruses that replicate in the cytoplasm of the infected host cell and induce modifications to host cell membranes that serve as sites for viral RNA synthesis. The goal of the proposed research program is to define the process of coronavirus replicase protein targeting and viral RNA synthesis during coronavirus infection, identify cellular interacting proteins, and test the requirements for cellular proteins and pathways in successful coronavirus replication. The results of these studies will define common pathways of coronavirus replication, identify viral and cellular targets for studies of viral virulence, attenuation, and interference with coronavirus infection.